Structure and sequence of the mitochondrial 20S rRNA and tRNA tyr gene of Paramecium primaurelia

The sequence and structure of the large (20s) mitochondrial (mt) rRNA gene and flanking regions from Paramecium primaurelia have been determined. The gene contains two regions of strong homology with other large mt rRNAs: one 44-base region near the 5' end and a 321-base region near the 3' end. Another region of strong homology to both ends of E. coli 23s RNA exists at loci consistent with these regions. The Paramecium gene appears to be 2204 bases in length and contains slightly more homology to E. coli rRNA than its mammalian or fungal counterparts. The gene, located about 1200 bp from the replicative terminal end of the linear mt DNA, is transcribed in the same polarity as replication. Previous R-looping studies detected no large introns within the gene. Here we describe sequences resembling degenerate rRNAs, one of which could represent a small intron. A tRNA tyr gene was found on the same DNA strand, 127 bp downstream from the large rRNA presumptive 3' end. The tRNA is flanked on both sides by short DNA regions of approximately 90% A + T content.     

Sequence of the gene for isoleucine tRNA1 and the surrounding region in a ribosomal RNA operon of Escherichia coli

A DNA fragment of about 2000 base pairs carrying the gene for tRNA(1) (Ile) has been cloned from a total Eco RI endonuclease digest of Escherichia coli DNA. Sequence analyses revealed that about the first 850 base pairs from one end of the fragment contain a nucleotide sequence corresponding to that in the 3'-end of 16S rRNA. The gene for tRNA(Ile) follows the 16S rRNA gene and both genes flank a spacer sequence of 68 base pairs. The spacer region contains a repeating, a hair pin and a symmetrical structure when the sequence is viewed in the single stranded form. A notable hair pin structure is also observed in the region adjacent to the 3'-end of the tRNA(1) (Ile) gene. In addition, about 850 base pairs from the other end of the DNA fragment have been found to contain the nucleotide sequence of the 5'-end of 23S rRNA. The presence of the genes for tRNA(1) (Ile), 16S and 23S rRNA and the hybridization to tRNA(1) (Ala) suggest that this cloned DNA is part of one of the E. coli rRNA operons carrying these two tRNA genes as a spacer.Images     

Mitochondrial gene order is not conserved in arthropods: prostriate and metastriate tick mitochondrial genomes

The entire mitochondrial genome was sequenced in a prostriate tick, Ixodes hexagonus, and a metastriate tick, Rhipicephalus sanguineus. Both genomes encode 22 tRNAs, 13 proteins, and two ribosomal RNAs. Prostriate ticks are basal members of Ixodidae and have the same gene order as Limulus polyphemus. In contrast, in R. sanguineus, a block of genes encoding NADH dehydrogenase subunit 1 (ND1), tRNA(Leu)(UUR), tRNA(Leu)(CUN), 16S rDNA, tRNA(Val), 12S rDNA, the control region, and the tRNA(Ile) and tRNA(Gln) have translocated to a position between the tRNA(Glu) and tRNA(Phe) genes. The tRNA(Cys) gene has translocated between the control region and the tRNA(Met) gene, and the tRNA(Leu)(CUN) gene has translocated between the tRNA(Ser)(UCN) gene and the control region. Furthermore, the control region is duplicated, and both copies undergo concerted evolution. Primers that flank these rearrangements confirm that this gene order is conserved in all metastriate ticks examined. Correspondence analysis of amino acid and codon use in the two ticks and in nine other arthropod mitochondrial genomes indicate a strong bias in R. sanguineus towards amino acids encoded by AT-rich codons.      

Numerous gene rearrangements in the mitochondrial genome of the wallaby louse, Heterodoxus macropus (Phthiraptera)

The complete arrangement of genes in the mitochondrial (mt) genome is known for 12 species of insects, and part of the gene arrangement in the mt genome is known for over 300 other species of insects. The arrangement of genes in the mt genome is very conserved in insects studied, since all of the protein-coding and rRNA genes and most of the tRNA genes are arranged in the same way. We sequenced the entire mt genome of the wallaby louse, Heterodoxus macropus, which is 14,670 bp long and has the 37 genes typical of animals and some noncoding regions. The largest noncoding region is 73 bp long (93% A+T), and the second largest is 47 bp long (92% A+T). Both of these noncoding regions seem to be able to form stem-loop structures. The arrangement of genes in the mt genome of this louse is unlike that of any other animal studied. All tRNA genes have moved and/or inverted relative to the ancestral gene arrangement of insects, which is present in the fruit fly Drosophila yakuba. At least nine protein-coding genes (atp6, atp8, cox2, cob, nad1-nad3, nad5, and nad6) have moved; moreover, four of these genes (atp6, atp8, nad1, and nad3) have inverted. The large number of gene rearrangements in the mt genome of H. macropus is unprecedented for an arthropod.     

Complete nucleotide sequences of mitochondrial genomes of two solitary entoprocts, Loxocorone allax and Loxosomella aloxiata: implications for lophotrochozoan phylogeny

The complete nucleotide sequences of the mitochondrial (mt) genomes of the entoprocts Loxocorone allax and Loxosomella aloxiata were determined. Both species carry the typical gene set of metazoan mt genomes and have similar organizations of their mt genes. However, they show differences in the positions of two tRNA(Leu) genes. Additionally, the tRNA(Val) gene, and half of the long non-coding region, is duplicated and inverted in the Loxos. aloxiata mt genome. The initiation codon of the Loxos. aloxiata cytochrome oxidase subunit I gene is expected to be ACG rather than AUG. The mt gene organizations in these two entoproct species most closely resemble those of mollusks such as Katharina tunicata and Octopus vulgaris, which have the most evolutionarily conserved mt gene organization reported to date in mollusks. Analyses of the mt gene organization in the lophotrochozoan phyla (Annelida, Brachiopoda, Echiura, Entoprocta, Mollusca, Nemertea, and Phoronida) suggested a close phylogenetic relationship between Brachiopoda, Annelida, and Echiura. However, Phoronida was excluded from this grouping. Molecular phylogenetic analyses based on the sequences of mt protein-coding genes suggested a possible close relationship between Entoprocta and Phoronida, and a close relationship among Brachiopoda, Annelida, and Echiura.     

Mutations in mitochondrial tRNA genes: non-linkage with syndromes of Wolfram and chronic progressive external ophthalmoplegia.

We have recently identified a point mutation in the mitochondrially encoded tRNA(Leu(UUR)) gene which associates with a combination of type II diabetes mellitus and sensorineural hearing loss in a large pedigree. To extend this finding to other syndromes which exhibit a combination of diabetes mellitus and hearing loss we have sequenced all mitochondrial tRNA genes from two patients with the Wolfram syndrome, a rare congenital disease characterized by diabetes mellitus, deafness, diabetes insipidus and optic atrophy. In each patient, a single different mutation was identified. One is an A to G transition mutation at np 12,308 in tRNA(Leu(CUN)) gene in a region which is highly conserved between species during evolution. This mutation has been described by Lauber et al. (1) as associating with chronic progressive external ophthalmoplegia (CPEO). The other is a C to T transition mutation at np 15,904 in tRNA(Thr) gene. Both mutations are also present in the general population (frequency tRNA(Leu(CUN)) mutation 0.16, tRNA(Thr) mutation 0.015). These findings suggest that evolutionarily conserved regions in mitochondrial tRNA genes can exhibit a significant polymorphism in humans, and that the mutation at np 12,308 in the tRNA(Leu(CUN)) gene is unlikely to be associated with CPEO and Wolfram syndrome.     

Combinatorial analysis of loop nucleotides in human mitochondrial tRNALeu(UUR)

A series of disease-related mutations are known to affect the hs mt tRNA(Leu(UUR)) gene, and the molecular-level properties of this tRNA may underlie the effects of pathogenic sequence changes. A combinatorial approach has been used to explore the importance of the D, TPsiC, and anticodon loops of hs mt tRNA(Leu(UUR)) in the structure and function of this molecule. A tRNA library was constructed with 20 randomized nucleotides in the loop regions of hs mt tRNA(Leu(UUR)), and tRNA variants that were aminoacylated by hs mt LeuRS were isolated using an in vitro selection approach. Analysis of 26 selected sequences revealed that a stabilized anticodon stem significantly enhances aminoacylation activity. However, anticodon loop nucleotides were not conserved in the active sequences, indicating that this region of hs mt tRNA(Leu(UUR)) is not involved in recognition by LeuRS. Within the D and TPsiC loops, only two nucleotides conserved their identities, while new sequences were selected that likely mediate interloop interactions. The results indicate that hs mt tRNA(Leu(UUR)), which is known to have structurally weak D and anticodon stems, benefits functionally from the introduction of stabilizing interactions. However, the locations of individual nucleotides that govern discrimination of this tRNA by hs mt LeuRS still remain obscure.     

Analysis of DNA encoding 23S rRNA and 16S–23S rRNA intergenic spacer regions from Plesiomonas shigelloides

Amplification of the gene encoding 23S rRNA of Plesiomonas shigelloides by polymerase chain reaction (PCR), with primers complementary to conserved regions of 16S and the 3' end of 23S rRNA genes, resulted in a DNA fragment of approximately 3 kb. This fragment was cloned in Escherichia coli and its nucleotide sequence determined. The region encoding 23S rRNA shows high homology with the published sequences of 23S rRNA from other members of the gamma division of Proteobacteria. The sequence of the intergenic spacer region, between the 16S and 23S rRNA genes, was determined in a further two clones. In one the sequence of a single tRNA(Glu) was found which was absent from the other two. This variation in sequence suggests that the different clones may be derived from different ribosomal RNA operons.     

Mitochondrial diversity of early-branching metazoa is revealed by the complete mt genome of a haplosclerid demosponge

The first mitochondrial (mt) genomes of demosponges have recently been sequenced and appear to be markedly different from published eumetazoan mt genomes. Here we show that the mt genome of the haplosclerid demosponge Amphimedon queenslandica has features that it shares with both demosponges and eumetazoans. Although the A. queenslandica mt genome has typical demosponge features, including size, long noncoding regions, and bacterialike rRNA genes, it lacks atp9, which is found in the other demosponges sequenced to date. We found strong evidence of a recent transposon-mediated transfer of atp9 to the nuclear genome. In addition, A. queenslandica bears an incomplete tRNA set, unusual amino acid deletion patterns, and a putative control region. Furthermore, the arrangement of mt rRNA genes differs from that of other demosponges. These genes evolve at significantly higher rates than observed in other demosponges, similar to previously observed nuclear rRNA gene rates in other haplosclerid demosponges.     

Staphylococcus aureus has clustered tRNA genes.

The polymerase chain reaction (PCR) was used to detect large tRNA gene clusters in Bacillus subtilis, Bacillus badius, Bacillus megaterium, Lactobacillus brevis, Lactobacillus casei, and Staphylococcus aureus. The primers were based on conserved sequences of known gram-positive bacterial tRNA(Arg) and tRNA(Phe) genes. This PCR procedure detected an unusually large tRNA gene cluster in S. aureus. PCR-generated probes were used to identify a 4.5-kb EcoRI fragment that contained 27 tRNA genes immediately 3' to an rRNA operon. Some of these 27 tRNA genes are very similar, but only 1 is exactly repeated in the cluster. The 5' end of this cluster has a gene order similar to that found in the 9- and 21-tRNA gene clusters of B. subtilis. The 3' end of this S. aureus cluster exhibits more similarity to the 16-tRNA gene cluster of B. subtilis. The 24th, 25th, and 26th tRNA genes of this S. aureus tRNA gene cluster code for three similar, unusual Gly-tRNAs that may be used in the synthesis of the peptidoglycan in the cell wall but not in protein synthesis. Southern analysis of restriction digests of S. aureus DNA indicate that there are five to six rRNA operons in this bacterium's genome and that most or all may have large tRNA gene clusters at the 3' end.     

Mutations in mitochondrial tRNA genes: a frequent cause of neuromuscular diseases.

We have sequenced the tRNA genes of mtDNA from patients with chronic progressive external ophthalmoplegia (CPEO) without detectable mtDNA deletions. Four point mutations were identified, located within highly conserved regions of mitochondrial tRNA genes, namely tRNA(Leu)(UAG), tRNA(Ser)(GCU), tRNA(Gly) and tRNA(Lys). One of these mutations (tRNA(Leu)(UAG)) was found in four patients with different forms of mitochondrial myopathy. An accumulation of three different tRNA point mutations (tRNA(Leu)(UAG)), tRNA(Ser)(GCU) and tRNA(Gly) was observed in a single patient, suggesting that mitochondrial tRNA genes represent hotspots for point mutations causing neuromuscular diseases.     

The histidine tRNA genes of yeast

Yeast has at least seven nuclear histidine tRNA genes although there is a single tRNAHis. We have sequenced three of the histidine tRNA genes. The genes have identical coding sequences and the DNA anti-codon sequence GTG corresponds to the GUG anti-codon in tRNAHis. None of the three yeast histidine tRNA genes has an intervening sequence. Two of the three genes contain repeated DNA elements in the region adjacent to the 5' end of the histidine tRNA gene. One of the elements, sigma, is 18 base pairs (bp) from the 5' end of each of these genes, sigma elements are highly conserved and flanked by 5-bp repeats. The other element, delta, is at variable distances from the tRNA gene; one is 439 bp from a histidine tRNA gene and the other is 52 bp from a histidine tRNA gene. These solo delta elements are quite divergent when compared with delta s associated with transposon yeast elements and are not flanked by 5-bp repeats.     

Mitochondrial genomic comparisons of the subterranean termites from the Genus Reticulitermes (Insecta: Isoptera: Rhinotermitidae).

Termites of the genus Reticulitermes are some of the most significant pests of structural timber and tree farming in the northern hemisphere, causing losses in the billions of dollars annually because of direct damage and termite control costs. This group has been frequently targeted for population genetic, phylogenetic, and species limit studies, most of which use mitochondrial (mt) genes; however, only a small fraction of the genome has been sequenced. The entire mt genome was sequenced for the eastern North American members of Reticulitermes: R. flavipes, R. santonensis, R. virginicus, and R. hageni. The mt genome has the same gene content and organization as that found in most insect species; however, the nucleotide composition and skew are highly biased (AT% low, strong A- and C-skew). Both the protein-coding and transfer RNA genes show high absolute levels of nucleotide substitution, suggesting that the high rates of mutation within Reticulitermes inferred from analyses of single mt genes are a general characteristic of the entire mt genome. The AT-rich or control region has a remarkable structure not previously observed in insect mt genomes. The majority of the control region is made up of 2 sets of repeat units, typically with 2 full and 1 partial copies of both the A (or small; 186 bp) and B (or large; 552 bp) repeats. The partial repeat units overlap by 36 bp. The size, location, and degree of overlap for the partial repeat units correspond to highly conserved stem/loop structures within the repeat units, suggesting that these structures are involved in the replication-mediated processes that govern repeat-unit evolution within mt genomes. Finally, molecular variation within the mt gene regions was compared with previous regions used in molecular diagnostics or phylogenetics of Reticulitermes. High numbers of single nucleotide polymorphisms were found in each of the mt genes, and some of the highest variability was found in gene regions that have not previously been investigated in this group. The whole mt genome sequence can thus be used to predict useful regions for future investigation.     

Gene rearrangements in snake mitochondrial genomes: highly concerted evolution of control-region-like sequences duplicated and inserted into a tRNA gene cluster

Mitochondrial DNA (mtDNA) regions corresponding to two major tRNA gene clusters were amplified and sequenced for the Japanese pit viper, himehabu. In one of these clusters, which in most vertebrates characterized to date contains three tightly connected genes for tRNA(Ile), and tRNA(Gln), and tRNA(Met), a sequence of approximately 1.3 kb was found to be inserted between the genes for tRNA(Ile) and tRNA(Gln). The insert consists of a control-region-like sequence possessing some conserved sequence blocks, and short flanking sequences which may be folded into tRNA(Pro), tRNA(Phe), and tRNA(Leu) genes. Several other snakes belonging to different families were also found to possess a control-region-like sequence and tRNA(Leu) gene between the tRNA(Ile)and tRNA(Gln) genes. We also sequenced a region surrounded by genes for cytochrome b and 12S rRNA, where the control region and genes for tRNA(Pro) and tRNA(Phe) are normally located in the mtDNAs of most vertebrates. In this region of three examined snakes, a control-region- like sequence exists that is almost completely identical to the one found between the tRNA(Ile) and tRNA(Gln) genes. The mtDNAs of these snakes thus possess two nearly identical control-region-like sequences which are otherwise divergent to a large extent between the species. These results suggest that the duplicate state of the control-region- like sequences has long persisted in snake mtDNAs, possibly since the original insertion of the control-region-like sequence and tRNA(Leu) gene into the tRNA gene cluster, which occurred in the early stage of the divergence of snakes. It is also suggested that the duplicated control-region-like sequences at two distant locations of mtDNA have evolved concertedly by a mechanism such as frequent gene conversion. The secondary structures of the determined tRNA genes point to the operation of simplification pressure on the T psi C arm of snake mitochondrial tRNAs.